Exosome-mediated diagnosis of hepatitis virus infections and diseases

ABSTRACT

A method for diagnosing hepatitis virus infection or a hepatitis disease condition in a subject based on hepatitis virus-associated biomarkers present on exosomes in a bodily fluid sample from the subject is disclosed. Also disclosed are a method for monitoring the course of a hepatitis virus infection or a hepatitis disease condition in a subject and a method for monitoring effectiveness of treatment to a subject with an anti-hepatitis virus agent based on hepatitis virus-associated biomarkers present on exosomes in bodily fluid samples from the subject, as well as a kit for diagnosing hepatitis virus infection and/or a hepatitis disease condition in a subject based on hepatitis virus-associated biomarkers on exosomes in bodily fluid samples from the subject.

This application is a continuation application of U.S. patentapplication Ser. No. 15/270,848, filed on Sep. 20, 2016, which is acontinuation application of U.S. patent application Ser. No. 14/159,612,filed on Jan. 21, 2014, which is continuation-in-part application ofU.S. patent application Ser. No. 12/572,652, filed Oct. 2, 2009, whichclaims priority from U.S. Provisional Application Ser. No. 61/102,941,filed Oct. 6, 2008. The entirety of all of the aforementionedapplications is incorporated herein by reference.

FIELD

The present invention generally relates to methods for diagnosis and, inparticular, to methods for diagnosing infections using biomarkerstargeting exosomes secreted in bodily fluids.

BACKGROUND

Exosomes are small vesicles 40-100 nm in diameter, that are secreted bya number of different cell types for communicating with other cells viathe proteins and ribonucleic acids they carry. An exosome is createdintracellularly when a segment of the cell membrane spontaneouslyinvaginates and is endocytosed. The internalized segment is broken intosmaller vesicles that are subsequently expelled from the cell. Thelatter stage occurs when the late endosome, containing many smallvesicles, fuses with the cell membrane, triggering the release of thevesicles from the cell. The vesicles (once released are called exosomes)consist of a lipid raft embedded with ligands common to the originalcell membrane.

Depending on their cellular origin, exosomes carry uniquely distinctprofiles of proteins and/or nucleic acids (such as microRNAs (miRNAs)),which can trigger signaling pathways in other cells and/or transferexosomal products into other cells by exosomal fusion with cellularplasma membranes. The protein composition of exosomes is distinct fromthat of other organelles, including early endosomes and plasmamembranes, more closely resembling that of late endosomes ormultivesicular bodies, (MVBs).

Exosome are released from different cell types in varied physiologicalcontexts. For example, B lymphocytes release exosomes carrying class IImajor histocompatibility complex molecules, which play a role inantigenic presentation. Similarly, dendritic cells produce exosomes(i.e., dexosomes, Dex), which play a role in immune response mediation,particularly in cytotoxic T lymphocyte stimulation. Some tumor cellssecrete specific exosomes (i.e., texosomes, Tex) carrying tumor antigensin a regulated manner, which can present these antigens to antigenpresenting cells. Exosomes may also carry pathogen-associated products.For example, exosomes have been known to carry products derived fromMycobacterium tuberculosis and Toxoplasma gondii-infected cells.

HIV and hepatitis virus infections are often assayed using serum orplasma. The detection of a specific viral antibody is presumptiveevidence of a corresponding viral infection, and is typically confirmedby the Western blot procedure. For example, detection of HIV virus byp24 antigen determination or detection of viral RNA by RT-PCR is alsoused to determine the amount of virus in circulation. CD4/CD8 T cellratios and other immune function tests are often used to monitor immunestatus and progression to AIDS. More recently, HIV tests using saliva orepithelia cells in the mouth have also been developed. However,currently there are few tests available to measure viral antigens orantibodies in urine. The detection of HIV and hepatitis proteins in theurine may provide a more rapid method to detect HIV or hepatitis virusinfections and/or monitor the progression of disease, particularlyviral-associated renal complications.

Hepatitis is an inflammation of the liver, most commonly caused by aviral infection. There are five main hepatitis viruses, referred to astypes A, B, C, D and E. Hepatitis A and E are typically caused byingestion of contaminated food or water. Hepatitis B, C and D usuallyoccur as a result of parenteral contact with infected body fluids (e.g.,from blood transfusions or invasive medical procedures usingcontaminated equipment). Hepatitis B is also transmitted by sexualcontact.

Hepatitis A virus (HAV) is an enterically transmitted viral disease thatcauses fever, malaise, anorexia, nausea, abdominal discomfort andjaundice. HAV is normally acquired by fecal-oral route, by eitherperson-to-person contact, ingestion of contaminated food or water ortransmission by pooled plasma products. The absence of a lipid envelopemakes HAV very resistant to physicochemical inactivation, and the viruscan withstand conventional heat treatment of blood products. Thedevelopment of sensitive and specific diagnostic assays to identify HAVantigens and/or antibodies in infected individuals as well as nucleicacid-based tests to detect viremic samples to exclude them fromtransfusion represents an important public health challenge.

Hepatitis B virus (HBV) infects humans and may result in two clinicaloutcomes. In the majority of clinical infections in adults (90-95%), thevirus is cleared after several weeks or months, and the patient developsa lifelong immunity against re-infection. In the remaining cases,however, the virus is not eliminated from the tissues, and the patientremains chronically infected. The sequelae of chronic infection areserious: such individuals are highly likely to develop scarring of theliver tissue (cirrhosis) and may eventually develop hepatocellularcarcinoma. HBV is transmitted via infected blood or other body fluids,especially saliva and semen, during delivery, sexual activity, orsharing of needles contaminated by infected blood.

Worldwide, it is estimated that 400 million people are chronicallyinfected with hepatitis B virus (HBV). Chronic hepatitis B (CHB)infection is the most common cause of liver cirrhosis and hepatocellularcarcinoma (HCC), with an estimated 500,000-900,000 deaths per year.Continuing HBV replication increases the risk of progression tocirrhosis and HCC.

Hepatitis C virus (HCV) is the causal agent for a largely chronic liverinfection originally identified as non-A, non-B hepatitis. HCV hasinfected about four million people in the United States and 170 millionworldwide, about four times as many as HIV and accounts for 90 to 95% ofthe hepatitis attributable to blood transfusion. It is presumed that theprimary route of infection is through contact with contaminated bodilyfluids, especially blood, from infected individuals. HCV infection isone of the primary causes of liver transplantation in the United Statesand other countries. Approximately 40-50% of the liver transplants inthe United States are based on HCV infections. The disease frequentlyprogresses to chronic liver damage. While the pathology of HCV infectionaffects mainly the liver, the virus is found in other cell types in thebody including peripheral blood lymphocytes.

The hepatitis delta virus (HDV) is a satellite RNA virus dependent onhepatitis B surface antigens to assemble its envelope and form newvirions to propagate infection. HDV has a small 1.7 Kb genome making itthe smallest known human virus. However, HDV is the most severe form ofviral hepatitis. Compared with other agents of viral hepatitis, acuteHDV infection is more often associated with fulminant hepatitis, arapidly progressive, often fatal form of the disease in which massiveamounts of the liver are destroyed. Chronic type D hepatitis istypically characterized by necroinflammatory lesions, similar to chronicHBV infection, but is more severe, and frequently progresses rapidly tocirrhosis and liver failure, accounting for the disproportionateassociation of chronic HDV infection with terminal liver disease.Although HDV infection affects fewer individuals than HBV alone, theresulting acute or chronic liver failure is a common indication forliver transplantation in Europe as well as North America. Chronic HDVdisease affects 15 million persons worldwide, about 70,000 of whom arein the U.S. The Centers for Disease Control estimates 1,000 deathsannually in the U.S. due to HDV infection.

In view of the wide scope of individuals affected by infectious agents,including various HIV and hepatitis-virus isolates, and the lack ofreliable, rapid, cost-effective and less invasive diagnostic tests,there is a need for diagnostic tests for diagnosing infectious agentsand infectious disease conditions that is reliable, rapid,cost-effective and less invasive.

SUMMARY

One aspect of the present application relates to a method for diagnosinghepatitis virus infection or a hepatitis disease condition in a subjectcaused by a hepatitis virus, comprising: (a) preparation of an exosomefrom a bodily fluid sample from a subject; (b) contacting said exosomepreparation with one or more hepatitis virus-associated biomarkerbinding agent(s) selective for a hepatitis virus and/or with one or moredetection reagent(s) suitable for detecting one or more hepatitisvirus-associated biomarker(s); and (c) determining whether the exosomepreparation comprises at least one hepatitis virus biomarker, wherein adetermination of the presence of the at least one hepatitis virusbiomarker in step (c) is indicative of hepatitis virus infection orhepatitis disease condition in the subject and wherein a determinationof the absence of the at least one hepatitis virus biomarker in step (c)is indicative of the absence of hepatitis virus infection or a hepatitisdisease condition in the subject.

In a preferred embodiment, the bodily fluid is urine.

In some embodiments, the exosome preparation comprises whole exosomes.In other embodiments, the exosome preparation comprises an exosomelysate.

In one embodiment, the hepatitis virus is HAV. In another embodiment,the hepatitis virus is HBV. In a further embodiment, the hepatitis virusis HCV. In another embodiment, the hepatitis virus is HDV. In yetanother embodiment, the hepatitis virus is HEV.

In certain embodiments, the contacting step (b) further comprisescontacting each one of a plurality of exosome preparations with one ormore different hepatitis virus biomarker binding agent(s). In certainembodiments, each hepatitis virus biomarker binding agent is selectivefor a common hepatitis virus. In other embodiments, each hepatitis virusbiomarker binding agent is selective for a different hepatitis virus. Incertain particular embodiments, the biomarker binding agents areselective for HAV, HBV, HCV, HDV and HEV.

In some embodiments, step (b) comprises contacting the exosomepreparation with one or more detection reagents capable of detecting oneor more hepatitis virus-associated biomarker(s), whereby the detectionreagents are selected from the group consisting of one or more reagentsfor reverse transcription of RNAs, one or more PCR reagents foramplification and detection of genomic nucleic acids or mRNAs and one ormore oligonucleotides for detecting miRNAs.

In certain embodiments, step (b) comprises contacting said exosomepreparation with one or more hepatitis virus-associated biomarkerbinding agent(s) and one or more detection reagent(s) suitable fordetecting one or more hepatitis virus-associated biomarkers, wherein theone or more detection reagents are suitable for detecting a miRNAselected from the group consisting of miR-92a, miR-122, miR-148a,miR-194, miR-155, miR-483-5p and miR-671-5p, miR-106b, miR-1274a,miR-130b, miR-140-3p, miR-151-3p, miR-181a, miR-19b, miR-21, miR-24,miR-375, miR-5481, miR-93, and miR-941, miRNA-1, miRNA-122, miR-584,miR-517c, miR-378, miR-520f, miR-142-5p, miR-451, miR-518d, miR-215,miR-376a, miR-133b, miR-367 and combinations thereof.

In other embodiments, step (b) comprises contacting one or more exosomepreparation with one or more agents capable of detecting liver damageand/or hepatocellular carcinoma selected from the group consisting ofCD10, CD26, CD81, AST, ALT, α-fetoprotein (AFP) and its variousisoforms, including AFP-L1, AFP-L2, AFP-L3, AFP-P4, AFP-P5 (E-PHA), andmonosialylated AFP; des-carboxyprothrombin (DCP), α-1-fucosidase (AFU),γ-glutamyl transferase, glypican-3 (GPC-3), squamous cell carcinomaantigen (SCCA), golgi protein 73 (GP73) and mucin 1 (MUC-1), 14-3-3gamma, alpha-1-fucosidase, gamma-glutamyl transferase, glypican-3,squamous cell carcinoma antigen, protein C (PROC), retinal bindingprotein 4 (RBP4), alpha-1-B glycoprotein (A1BG), alpha-1-acidglycoprotein (AGP), Mac-2-binding protein (M2BP), complement Factor H(CFH), insulin-like growth factor binding protein acid labeled subunit(IGFALS) and combinations thereof.

The exosome preparation may be prepared by any exosome isolationprocedure including differential centrifugation, nanomembraneultrafiltration, immunoabsorbent capture, size-exclusion chromatography,ultracentrifugation, magnetic activated cell sorting (MACS) andcombinations thereof.

In certain embodiments, the diagnosis step is further coupled with thestep of administering to the subject a therapeutic drug for hepatitis orthe step of obtaining a liver biopsy from the subject for analysis. Anyconventional therapeutic drug for hepatitis may be employed.

Another aspect of the present application relates to a method formonitoring the course of a hepatitis virus infection or a hepatitisdisease condition in a subject. The method comprises: (a) measuring thelevel of one or more hepatitis virus-associated biomarkers in a firstexosome preparation representing a first time point from a subject; (b)measuring the level of the one or more hepatitis virus-associatedbiomarkers in a second exosome preparation representing a second timepoint from a subject; (c) comparing the level of the one or morehepatitis virus-associated biomarkers in the first exosome preparationto the level of the one or more hepatitis virus-associated biomarkers inthe second exosome preparation; and (d) determining the diseaseprogression between the first time point and the second time point basedon the result of step (c).

Another aspect of the present application relates to a method formonitoring the effectiveness of treatment to a subject with ananti-hepatitis virus agent. The method comprises: (a) determining ahepatitis virus-associated biomarker profile in a first exosomepreparation from a subject prior to administration of an anti-hepatitisvirus agent; (b) determining a hepatitis virus-associated biomarkerprofile in a second exosome preparation from said subject afteradministration of said anti-hepatitis virus agent; (c) comparing thehepatitis virus-associated biomarker profile in the first exosomepreparation with the hepatitis virus-associated biomarker profile in thesecond exosome preparation; and (d) determining the effectiveness of theanti-hepatitis virus agent based on a comparative analysis of thehepatitis virus-associated biomarker profiles in step (c).

Another aspect of the present application relates to a kit fordiagnosing hepatitis virus infection and/or a hepatitis diseasecondition in a subject. The kit comprises: one or more reagents forpreparation of an exosome(s); one or more hepatitis virus-associatedbiomarker binding agents and detection reagents selective for ahepatitis virus, and one or more hepatitis virus-associated biomarkerstandards.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing an embodiment of an exemplary method fordetecting HIV-infection or monitoring the progress of HIV-infection in asubject using a urine sample from the subject.

FIGS. 2A-2C are composites of samples SELDI-TOF-MS spectrum of urinaryexosomes from patients in the HIVAN groups.

FIGS. 3A-3D are composites of samples SELDI-TOF-MS spectrum of urinaryexosomes from patients in the AA HIV+ groups.

FIGS. 4A-4C are composites of samples SELDI-TOF-MS spectrum of urinaryexosomes from patients in the HIV White groups.

FIGS. 5A-5E are composites of samples SELDI-TOF-MS spectrum of urinaryexosomes from patients in the FSGS groups.

FIGS. 6A-6C are composites of samples SELDI-TOF-MS spectrum of urinaryexosomes from patients in the Normal Controls groups.

FIGS. 7A-7E are composites of transmission electron microscope (TEM)pictures of urinary exosomes isolated from patients from the HIVAN group(Figure A), the FSGS group (Figure B), the African American (AA) HIV+group (Figure C), the white HIV+ group (Figure D), and the normalcontrol group (Figure E).

FIG. 8 is a composite of pictures showing Western blot analysis ofurinary vesicles from HIV+ patients and controls. Vesicles were isolatedfrom urine by ultrafiltration and analyzed for the presence of HIV Nefor other HIV proteins. The top panel used anti-HIV Nef monoclonalantibodies, while the lower panel utilized pooled HIV+ patient sera sthe primary antibodies. Patients 27, 28, 30, 41 and 104 were AA.Patients 108, 103, 86 and 48 were HIV+ white patients. The last panel iscontrol panel for three HIV negative individuals, recombinant HIV Nefand p24.

FIG. 9 is a receiver operating characteristic (ROC) curve for hepatitisB diagnosis based on exosome ELISA.

FIG. 10 is a ROC curve for hepatitis C diagnosis based on exosome ELISA.

DETAILED DESCRIPTION

The practice of the embodiments described in further detail below willemploy, unless other wise indicated, conventional methods ofdiagnostics, molecular biology, cell biology, biochemistry andimmunology within the skill of the art. Such techniques are explainedfully in the literature. All publications, patents and patentapplications cited herein, whether supra or infra, are herebyincorporated by reference in their entirety.

It is appreciated that certain features of the invention, which are forclarity described in the context of separate embodiments may also beprovided in combination in a single embodiment. Conversely variousfeatures of the invention, which are for brevity, described in thecontext of a single embodiment, may also be provided separately and/orin any suitable sub-combination.

Definitions

As used herein, the following terms shall have the following meanings:

As used herein, the terms “biomarker” and “infectious agent-associatedbiomarker” are used interchangeably with reference to any molecularentity that can be used as an indicator of an acute infectious diseaseor chronic infectious disease condition in an organism. The biomarkermay be any detectable protein, nucleic acid, such as an mRNA ormicroRNA, lipid, or any product present and/or differentially expressedin exosomes present in bodily fluids following an infection and/orcoincident with an infectious disease condition whose presence and/orconcentration reflects the presence, severity, type or progression of anacute or chronic infection in a subject. In molecular terms, biomarkersmay be detected and quantitated in a subject using genomics, proteomicstechnologies or imaging technologies.

An infectious agent-associated biomarker may be viral, bacterial,fungal, protozoan in nature (i.e., encoded by a virus, bacteria, fungus,protozoan etc.) or it may be cellular in nature. Thus, an infectiousagent-associated biomarker may be directly derived from the infectiousagent, such that the infectious agent encodes for the biomarker. As usedherein, the term “virus biomarker” refers to a biomarker directlyderived or encoded for by the virus. For example, a hepatitis virusbiomarker refers a biomarker directly derived or encoded for by ahepatitis virus. The terms “infectious agent associated cellularbiomarker” and “virus-associated cellular biomarker” refer to cellularbiomarkers whose expression is altered in response to an infectiousagent (such as a virus) or infectious disease condition and whosedifferential expression relative to non-infected cells is diagnostic ofan infection or disease caused by that particular infectious agent.

As used herein, the term “gene product” or “expression product of agene” refers to the transcriptional products of a gene, such as mRNAsand cDNAs encoded by the gene, and/or the translational products of agene, such as peptides encoded by the gene, and fragments thereof.

As used herein, the term “infectious disease conditions” refers toconditions that are related to, or resulted from, an infectious disease.As used herein, the term “hepatitis disease conditions” include, but arenot limited to, hepatitis, cirrhosis and hepatocellular carcinoma (HCC).

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed.

One aspect of the present application relates to a method for diagnosingan infection or infectious disease related condition in a subject. Theinfection may be acute or chronic. In one embodiment, the methodcomprises the steps of (a) isolating exosomes from a bodily fluid sampleof said subject; (b) detecting the presence of one or more infectiousagent-associated biomarker(s) from the isolated exosomes; and (c)determining whether the isolated exosomes exhibit a biomarker profilecharacteristic of an individual who is acutely or chronically infectedwith a particular infectious agent.

The detecting step may be carried out using any methodology suitable foridentifying an infectious agent-associated biomarker, including but notlimited to one-dimensional and two-dimensional electrophoretic gelanalysis, electrophoresis, Western blot, HPLC, FPLC, ELISA, massspectrometry (MS), protein sequencing, nucleotide sequencing, PCR,antibody array and combinations thereof.

The determining step may be based on identifying the presence, absenceand/or altered expression profiles of one or more infectiousagent-associated biomarker(s) in the isolated exosomes obtained from abodily fluid sample. The determining step may carried out by comparingan infectious agent-associated biomarker profile in a bodily fluidsample (such as urine) to an infectious agent-associated biomarkerprofile stored in a database. A diagnosis may be based on the results ofthis comparison.

As used herein, the term “bodily fluid sample” refers to a sample ofbodily fluid obtained from a mammal subject, preferably a human subject.Exemplary bodily fluid samples include urine, blood, saliva, serum,plasma, cyst fluid, pleural fluid, ascites fluid, peritoneal fluid,amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolarlavage fluid, breast milk, tears, sputum, and combinations thereof. In apreferred embodiment, the bodily fluid sample is urine. Unless otherwisenoted, as used herein, the terms “bodily fluid sample” and “sample” areto be considered synonymous with any of the above-described bodily fluidsamples.

The biomarker profile may consist of one or more biomarkers directlyderived from the infectious agent and/or one or more cellular product(s)whose expression profile is characteristic of an individual who isacutely or chronically infected with a particular infectious agent.Accordingly, the step of determining whether the subject carries aninfectious agent may be based on detecting the presence, absence ordifferential expression of one or more infectious agent-associatedbiomarker(s) present in the isolated exosomes. As used herein, the term“differential expression” refers to a qualitative and/or quantitativechanges in biomarker expression levels relative to a control sample.

The term “increased level” refers to an expression level that is higherthan a normal or control level customarily defined or used in therelevant art. For example, an increased level of immunostaining of anexosome preparation from a bodily fluid sample is a level ofimmunostaining that would be considered higher than the level ofimmunostaining of a control exosome preparation by a person of ordinaryskill in the art. As used herein, the described biomarker may exhibitincreased expression levels of at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 80%, at least 100%, at least2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least50-fold or at least 100-fold increase or more relative to a suitablereference level.

The term “decreased level” refers to an expression level that is lowerthan a normal or control level customarily defined or used in therelevant art. As used herein, the described biomarkers may exhibitdecreased expression levels of at least at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 80%, at least 100%, atleast 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, atleast 50-fold or at least 100-fold decrease or more relative to asuitable reference level.

The term “expression level of an infectious agent-associated biomarker”may be measured at the transcription level, in which case the presenceand/or the amount of a polynucleotide is determined, or at thetranslation level, in which case the presence and/or the amount of apolypeptide is determined.

Infectious agent-associated biomarker expression levels may becharacterized using any suitable method. Expression levels andexpression ratios may be determined at the mRNA level (e.g., by RT-PCR,QT-PCR, oligonucleotide array, etc.) or at the protein level (e.g., byELISA, Western blot, antibody microarray, etc.). Preferred methodologiesfor determining mRNA or miRNA expression levels include quantitativereverse transcriptase PCR (QT-PCR), quantitative real-time RT-PCR,oligonucleotide microarray, antibody microarray, or combination thereof.Preferred methodologies for determining protein expression levelsinclude the use of ELISAs and antibody microarrays.

In certain embodiments, an infectious agent-associated biomarker profilemay contain from zero to multiple infectious agent-associatedbiomarkers. Thus, by way of example, an infectious agent-associatedprofile of a healthy subject may contain no infectious agent-associatedbiomarkers, whereas infectious agent-associated profile of a patientwith an infectious disease may contain a plurality of infectiousagent-associated biomarkers. In this method, the genetic background andpertinent information from the medical record of the subject may also beused in the determining step to make a diagnosis.

In another aspect, the method is used to monitor the progression of aninfectious disease or infectious disease related condition in thesubject based on the presence, absence and/or altered expressionprofiles of one or more infectious agent-associated biomarker(s) in theisolated exosomes obtained from a bodily fluid sample.

In a related aspect, the method is used to monitor the course of aninfectious disease or infectious disease related condition in a subjectcomprises the steps of (a) measuring the level of one or more biomarkersin exosomes of a first sample obtained from the subject at a first timepoint; (b) measuring the level of the one or more biomarkers in exosomesof a second sample obtained from the subject at a second time point; (c)comparing the level of the one or more biomarkers at the first timepoint to the level of the one or more biomarkers at the second timepoint; and (d) determining the disease progression between the first andthe second time point based on the result of step (c).

Another aspect of the present application relates to a method formonitoring the effectiveness of a therapeutic agent in a subject as afunction of infectious agent-associated biomarker levels present in theexosomes obtained from a bodily fluid sample. This method includes thesteps of: (a) determining an infectious agent-associated biomarkerprofile in the exosomes of a sample obtained from a subject prior toadministration of the therapeutic agent; (b) determining an infectiousagent-associated biomarker profile in the exosomes of one or moresamples obtained from the subject after administration of thetherapeutic agent; (c) comparing the infectious agent-associatedbiomarker profile in the pre-administration sample with the infectiousagent-associated biomarker profile in the post-administration sample(s);and (d) determining the effectiveness of the therapeutic agent based ona comparative analysis of the biomarker profiles in step (c).

In certain embodiments, the method may further contain the step ofaltering the administration of the agent to the subject. In accordancewith this method, the infectious agent-associated biomarker profile maybe used as an indicator of the effectiveness of an agent, even in theabsence of an observable phenotypic response.

Exemplary mammal subjects for use in accordance with the methodsdescribed herein include humans, monkeys, gorillas, baboons, anddomesticated animals, such as cows, pigs, horses, rabbits, dogs, cats,goats and the like.

Infectious Agents and Diseases

The infectious agent may be viral, bacterial or fungal in nature. In oneembodiment, the infectious agent is human immunodeficiency virus (HIV)type 1 or type 2 (HIV-1 and HIV-2). In another embodiment, theinfectious agent is a hepatitis virus selected from the group consistingof hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus(HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV).

In addition to the HIV and hepatitis viruses, additional virusesinclude, but are not limited to, human T-cell lymphotropic virus (HTLV)type I and type II (HTLV-I and HTLV-II), parvovirus B19 virus,transfusion transmitted virus (TTV); measles virus; rotaviruses,including Types A, B, C, D, and E; herpesviruses, including Epstein-Barrvirus, human cytomegalovirus type 1 (HCMV-1), herpes simplex virus (HSV)types 1 and 2 (HSV-1 and HSV-2), human herpes virus type 6 (HHV-6),human herpes virus type 7 (HHV-7), human herpes virus type 8 (HHV-8);human papilloma virus (HPV) and its many serotypes; influenza type Aviruses, including subtypes H1N1 and H5N1; severe acute respiratorysyndrome (SARS) coronavirus; and other miscellaneous RNA viruses,including Arenaviridae (e.g., Lassa fever virus (LFV)), Filoviridae(e.g., Ebola virus (EBOV) and Marburg virus (MBGV)); Bunyaviridae (e.g.,Rift Valley fever virus (RVFV) and Crimean-Congo hemorrhagic fever virus(CCHFV); and Flaviviridae, including West Nile virus (WNV), Dengue fevervirus (DENV), yellow fever virus (YFV), and GB virus C (GBV-C), formerlyknown as Hepatitis G virus (HGV).

Exemplary bacteria include, but are not limited to Mycobacteriumspecies, including M. tuberculosis; Staphylococcus species, including S.epidermidis, S. aureus, and methicillin-resistant S. aureus;Streptococcus species, including S. pneumoniae, S. pyogenes, S. mutans,S. agalactiae, S. equi, S. canis, S. bovis, S. equinus, S. anginosus, S.sanguis, S. salivarius, S. mitis; other pathogenic Streptococcalspecies, including Enterococcus species, such as E. faecalis and E.faecium; Haemophilus influenzae, Pseudomonas species, including P.aeruginosa, P. pseudomallei, and P. mallei; Salmonella species,including S. enterocolitis, S. typhimurium, S. enteritidis, S. bongori,and S. choleraesuis; Shigella species, including S. flexneri, S. sonnei,S. dysenteriae, and S. boydii; Brucella species, including B.melitensis, B. suis, B. abortus, and B. pertussis; Neisseria species,including N. meningitidis and N. gonorrhoeae; Escherichia coli,including enterotoxigenic E. coli (ETEC); Vibrio cholerae, Helicobacterpylori, Chlamydia trachomatis, Clostridium difficile, Cryptococcusneoformans, Moraxella species, including M. catarrhalis, Campylobacterspecies, including C. jejuni; Corynebacterium species, including C.diphtheriae, C. ulcerans, C. pseudotuberculosis, C.pseudodiphtheriticum, C. urealyticum, C. hemolyticum, C. equi; Listeriamonocytogenes, Nocardia asteroides, Bacteroides species, Actinomycetesspecies, Treponema pallidum, Leptospirosa species, Klebsiellapneumoniae; Proteus sp., including Proteus vulgaris; Serratia species,Acinetobacter, Yersinia species, including Y. pestis and Y.pseudotuberculosis; Francisella tularensis, Enterobacter species,Bacteroides species, Legionella species, Borrelia burgdorferi, and thelike.

Exemplary fungi include, but are not limited to, Aspergillus species,Dermatophytes, Blastomyces derinatitidis, Candida species, including C.albicans and C. krusei; Malassezia furfur, Exophiala werneckii, Piedraiahortai, Trichosporon beigelii, Pseudallescheria boydii, Madurellagrisea, Histoplasma capsulatum, Sporothrix schenckii, Histoplasmacapsulatum, Tinea species, including T. versicolor, T. pedis T. unguium,T. cruris, T. capitus, T. corporis, T. barbae; Trichophyton species,including T. rubrum, T. interdigitale, T. tonsurans, T. violaceum, T.yaoundei, T. schoenleinii, T. megninii, T. soudanense, T. equinum, T.erinacei, and T. verrucosum; Mycoplasma genitalia; Microsporum species,including M. audouini, M. ferrugineum, M. canis, M. nanum, M. distortum,M. gypseum, M. fulvum, and the like.

Exemplary protozoans include, but are not limited to Plasmodiumfalciparum, Cryptosporidium, Isospora belli, Toxoplasma gondii,Trichomonas vaginalis, and Cyclospora species.

An additional infectious agent includes the protease-resistant form ofthe prion protein (PrP), named scrapie disease associated prion protein(PrP^(Sc)), which is associated with a group of fatal neurodegenerativeinfectious pathologies, including the Creutzfeldt-Jakob disease (CJD),and is known to be associated with exosomes.

In certain embodiments, the infectious disease or infectious diseasecondition affects the kidney, such as pyelonephritis. In certainpreferred embodiments, the infectious disease related condition isHIV-associated nephropathy (HIVAN).

In other embodiments, the infectious disease or infectious diseasecondition affects the liver, such as hepatitis, cirrhosis andhepatocellular carcinoma (HCC).

Biomarkers

The infectious agent-associated biomarker may be viral, bacterial,fungal or protozoan in nature (i.e., encoded by a virus, bacteria,fungus, protozoan etc.). The infectious agent-associated biomarker maybe directly derived from the infectious agent. Alternatively, theinfectious agent-associated biomarker may be a cellular biomarkerproduct whose differential expression relative to non-infected cells isdiagnostic of an infection or disease caused by that particularinfectious agent.

In some embodiments, the biomarker is a protein. In other embodiments,the biomarker is a nucleic acid. Exemplary nucleic acids include bothsingle-stranded and double-stranded polynucleotides or oligonucleotidesof DNA or RNA. Exemplary nucleic acids include viral genomic DNAs orRNAs, including reverse transcribed derivatives thereof.

In certain embodiments, viral genomic nucleic acids associated withviral particles (such as hepatitis C virus (HCV)) contained in isolatedexosomes may be isolated by conventional RNA or DNA purificationmethodologies employing e.g., silica gel based spin columns forpurification of viral nucleic acids from cell-free body fluids (Qiagen).

In other embodiments, the biomarker is a viral messenger RNA (mRNA),viral microRNA (miRNA) or a viral-induced miRNA. Both mRNAs and miRNAsare known to be shuttled through exosomes. MicroRNAs are smallnon-coding RNAs responsible of post-transcriptional regulation of geneexpression through interaction with messenger RNAs (mRNAs). They areinvolved in important biological processes and are often dysregulated ina variety of diseases, including cancer and infections. Viruses alsoencode their own miRNAs, which can be loaded into RNA-induced silencingcomplexes (RISC) for gene silencing of host's genes and/or their own viablocking mRNA translation and/or initiating mRNA decay. In the past fewyears evidence of the presence of cellular miRNAs in extracellular humanbody fluids such as serum, plasma, saliva, and urine has accumulated,including their cofractionation (or colocalization) with exosomes.

Exemplary HIV-1-associated miRNAs include hiv1-mir-H1 andhiv-1-miR-N367.

Exemplary hepatitis-associated miRNAs include miR-122 and mrR-199.

Preferred infectious agent-associated biomarkers include, but are notlimited to HIV (e.g., HIV-1, HIV-2)-associated biomarkers and hepatitisvirus (e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitisC virus (HCV), hepatitis D virus (HDV) and hepatitis E virus(HEV))-associated biomarkers. As used herein, the terms “HIV-associatedbiomarker” or “hepatitis virus-associated biomarker” refer to HIV orhepatitis virus proteins, nucleic acids or fragments thereof, as well ascellular biomarker products whose differential expression relative tonon-infected cells is diagnostic of HIV or hepatitis virus infections.

In certain particular embodiments, the biomarker is an HIV-associatedprotein selected from the group consisting of Nef, gp120, protease, Vif,Gag-Pol, Gag, p24, Rev, reverse transcriptase (RT), Tat, p1, p17, Vpu,Vpr, gp41 and DNA polymerase.

Hepatitis Virus-Associated Biomarkers

In other embodiments the biomarker is a hepatitis virus biomarkerassociated with hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis D virus (HDV) and/or hepatitis Evirus (HEV). Any of the hepatitis virus proteins or nucleic acidsdescribed herein may be utilized as hepatitis virus biomarkers orhepatitis virus-associated biomarkers in accordance with the presentapplication.

Hepatitis A virus (HAV) is a small, nonenveloped, spherical virusclassified in the genus Hepatovirus of the Picornaviridae family. TheHAV genome consists of a single-strand, linear, 7.5 kb RNA moleculeencoding a polyprotein precursor that is processed to yield thestructural proteins and enzymatic activities required for viralreplication. HAV encodes four capsid proteins (A, B, C and D) whichcontain the major antigenic domains recognized by antibodies of infectedindividuals. In addition to the capsid proteins, antigenic domains havebeen reported in nonstructural proteins such as 2A and the viral encodedprotease. Another important HAV antigenic domain has been described inthe junction between the capsid precursor P1 and 2A. In someembodiments, the HAV polyproteins VP0, VP1, and VP3 (aka 1AB, 1D, and1C, respectively) are used as HAV biomarkers.

Hepatitis B virus is an enveloped non-cytopathic double-strandedcircular DNA virus. It is a member of the Hepadnaviridae family. Thevirus consists of a central core that contains a core antigen (HBcAg)surrounded by an envelope containing a surface protein/surface antigen(HBsAg) and is 42 nm in diameter. It also contains an e antigen (HBeAg)which, along with HBcAg and HBsAg, is helpful in identifying thisdisease. In HBV virions, the genome is found in an incompletedouble-stranded form. Upon infection by HBV, the incomplete partialdouble stranded DNA is repaired to form a 3.2-kb cccDNA, which serves asa template to transcribe overlapping RNA species including a 3.5-kbpregenomic RNA coding for reverse-transcriptase (polymerase), core,PreS, S and X proteins. These RNAs are then translated into HBV proteinsor reverse-transcribed into HBV DNA. All of the HBV proteins playimportant roles in HBV transcriptional regulation, viral package,reverse-transcription and viral DNA recycling.

Exemplary hepatitis B virus (HBV) biomarker proteins for use in presentapplication include the HBV core antigen (HBcAg), HBV surface antigen(HBsAg), HBV e antigen (HBeAg), HBV X protein (HBx), HBV polymerase, andthe HBV envelope proteins S, M, and L.

HCV is an RNA virus of the Flaviviridae, genus Hepacivirus, and is mostclosely related to the pestiviruses, BVDV and GBV-B. The HCV genome iscomposed of a single positive strand of RNA, approximately 9.6 kb inlength. The HCV genome possesses a continuous, translational openreading frame (ORF) that encodes a polyprotein of about 3,000 aminoacids. The structural protein(s) appear to be encoded in approximatelythe first quarter of the N-terminus region of the ORF, the remaindercoding for non-structural proteins. The polyprotein serves as theprecursor to at least 10 separate viral proteins critical forreplication and assembly of progeny viral particles. The organization ofstructural and non-structural proteins in the HCV polyprotein is asfollows: C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b. Examples of HCVbiomarkers include, but are not limited to, HCV core antigen (HCVcAg),HCV C protein, HCV E1 protein, HCV E2 protein, HCV p7 protein, HCV NS2protein, HCV NS3 protein, HCV NS4a protein, HCV NS4b protein, HCV NS5aprotein and HCV NS5b protein.

The hepatitis delta virus (HDV) is a satellite RNA virus dependent onhepatitis B surface antigens to assemble its envelope and form newvirions to propagate infection. HDV has a small 1.7 Kb genome making itthe smallest known human virus. The HDV virion is composed of aribonucleoprotein core and an envelope. The core contains HDV-RNA, andhepatitis delta antigen (HDAg), which is the only protein encoded bythis virus. The envelope is formed by the surface antigen protein(hepatitis B surface antigen, or HBsAg) of the helper virus, hepatitisB. The envelope is the sole helper function provided by HBV. HDV is ableto replicate its RNA within cells in the absence of HBV, but requiresHBsAg for packaging and release of HDV virions, as well as forinfectivity. As a result of the dependence of HDV on HBV, HDV infectsindividuals only in association with HBV.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a form ofacute viral hepatitis that is endemic to many resource-limited regionsof the world. It is estimated that about 2 billion people, which isabout a third of the world population, live in areas endemic for HEV andare at risk for infection. In these areas, hepatitis E is the major formof acute hepatitis; in India for example about 50% of acute hepatitis isdue to HEV.

HEV is a small non-enveloped virus with a size of 27-34 nm and isclassified as a Hepevirus in the family Hepeviridae. The HEV genome is asingle-stranded RNA of −7.2 kb that is positive-sense, with a5′-methylguanine cap and a 3′ poly(A) stretch, and contains threepartially overlapping open reading frames (ORFs)—called orf1, orf2 andorf3. HEV orf1, a polyprotein of 1693 amino acids, encodes the viralnonstructural functions. Functional domains identified in the HEVnonstructural polyprotein include (starting from the N-terminalend)-methyltransferase (MeT), papain-like cysteine protease (PCP), RNAhelicase (Hel) and RNA dependent RNA polymerase (RdRp). HEV orf2 encodesa viral capsid protein of 660 amino acids, which is believed toencapsidate the viral RNA genome. HEV orf3 is believed to express a 114amino acid protein that is dispensable for replication in vitro and isbelieved to function as a viral accessory protein, likely affecting thehost response to infection.

Exemplary hepatitis viral nucleic acid sequences include, but are notlimited to, nucleic acid sequences involved in transcription andtranslation (e.g., En1, En2, X, P) and nucleic acid sequences encodingstructural proteins (e.g., core proteins including C and C-relatedproteins, capsid and envelope proteins including S, M, and/or Lproteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, 2001,supra). Exemplary hepatitis C nucleic acid sequences include, but arenot limited to, serine proteases (e.g., NS3/NS4), helicases (e.g., NS3),polymerases (e.g., NS5B), and envelope proteins (e.g., E1, E2, and p7).Hepatitis A nucleic acid sequences are set forth in, e.g., GenbankAccession No. NC_001489; hepatitis B nucleic acid sequences are setforth in, e.g., Genbank Accession No. NC_003977; hepatitis C nucleicacid sequences are set forth in, e.g., Genbank Accession No. NC_004102;hepatitis D nucleic acid sequence are set forth in, e.g., GenbankAccession No. NC_001653; hepatitis E nucleic acid sequences are setforth in, e.g., Genbank Accession No. NC_001434; and hepatitis G nucleicacid sequences are set forth in, e.g., Genbank Accession No. NC_001710.

Cellular Exosomal Biomarkers

Because of their cellular origin, exosomes bear specific protein markersof the endosomal pathway, such as tetraspanins (CD63, CD9 and CD81),heat shock proteins (HSP70) and proteins from the Rab family (e.g.,Rab5), Tsg101 and Alix, which are not found in other types of vesiclesof similar size. The composition of exosomes is also known to reflectthe particular cell types from which they are derived. Accordingly,exosome profiling using cellular exosome markers can provide informationcorrelating the presence of infectious agent biomarkers or infectiousagent associated cellular biomarkers with particular cell types in thecontext of diagnosing a disease. In addition, cellular exosomal surfacemarkers can provide useful targets for affinity purification/detectionof exosomes as further described below.

Cellular exosomal markers can provide useful internal controls fordetermining expression level changes relative to reference samples andcan provide useful markers helpful in diagnosing phenotypic tissuechanges, including e.g., liver damage, liver fibrosis, inflammation,hepatocellular carcinoma etc. Non-limiting examples of normal exosomemarker includes CD9, CD10, CD26, CD53, CD63, CD81, CD82, Rab5, Alix,TSG101, Hsc70 and Hsp90.

In certain embodiments, cellular exosomal markers reflecting tissuedamage and/or disease may be exclusively detected in a particulardisease state, such as hepatic fibrosis or hepatocellular carcinoma. Inother embodiments, the cellular exosomal markers may be increasedrelative to reference control samples or decreased relative to referencecontrol cells. The increases or decreases may be evident in terms oftotal expression levels in an exosomal preparation as a whole.Alternatively, the expression level changes may be evident in the totalnumber of exosomes that are positive or negative for a particularmarker.

Both types of liver epithelia (i.e., hepatocytes and cholangiocytes),natural killer T (NKT) cells, hepatic stellate cells, adult liver stemcells, and hepatic sinusoidal endothelial cells are exosome-releasingand/or exosome-targeting cells. Exosomal biomarkers associated withhepatic tissues and liver diseases are of particular interest, becausetheir markers can shed light in the incidence of prognosis ofhepatitis-related conditions, including hepatocellular carcinoma (HCC).

Like other cell-derived exosomes, hepatic-derived exosomes includetypical exosomal markers, which may be utilized in the presentapplication. These include common “marker” proteins, such astetraspanins (e.g., CD9, CD10, CD26, CD53, CD63, CD81, CD82);endosome-associated proteins that are involved in MVB biogenesis, suchas Alix and TSG101; cytoplasmic heat shock proteins, such as Hsc70 andHsp90; and hepatic cell-type specific proteins and nucleic acids,including mRNAs, microRNAs (miRNAs) and other non-coding RNAs, thecomposition of which depends on the functional state of the cells (e.g.,rested, stimulated, stressed, transformed, etc.)(Masyuk et al., J.Hepatol., 59(3):2013).

Liver Disease Markers

There are a number of markers that may be used individually or incombination for diagnosis of liver diseases, such as hepatitis, hepaticfibrosis and hepatocellular carcinoma. Exemplary liver disease markersinclude, but are not limited to CD10, CD26, CD81, AST, ALT,α-fetoprotein (AFP) and its various isoforms, including AFP-L1, AFP-L2,AFP-L3, AFP-P4, AFP-P5 (E-PHA), and monosialylated AFP;des-carboxyprothrombin (DCP), α-1-fucosidase (AFU), γ-glutamyltransferase, glypican-3 (GPC-3), squamous cell carcinoma antigen (SCCA),golgi protein 73 (GP73) and mucin 1 (MUC-1), 14-3-3 gamma,alpha-1-fucosidase, gamma-glutamyl transferase, glypican-3, squamouscell carcinoma antigen, protein C (PROC), retinal binding protein 4(RBP4), alpha-1-B glycoprotein (A1BG), alpha-1-acid glycoprotein (AGP),Mac-2-binding protein (M2BP), complement Factor H (CFH), insulin-likegrowth factor binding protein acid labeled subunit (IGFALS).

In certain embodiments, an increased level of A1BG or a decreased levelof CFH or IGFALS is/are indicative of the incidence and/or severity ofacute or chronic hepatitis. In other embodiments, decreasing levels ofprotein C (PROC) and/or retinal binding protein 4 (RBP4) are indicativeof the increasing severity of fibrosis.

miRNA markers for liver disease, such as liver fibrosis include miR-92a,miR-122, miR-148a, miR-194, miR-155, miR-483-5p and miR-671-5p, whichexhibit progressive increases at higher fibrotic stages and miR-106b,miR-1274a, miR-130b, miR-140-3p, miR-151-3p, miR-181a, miR-19b, miR-21,miR-24, miR-375, miR-5481, miR-93, and miR-941, which exhibitprogressive decreases in expression at higher fibrotic stages.

HCC cell-derived exosomes are known to contain an enriched fraction ofsmall RNAs. Exemplary HCC-associated miRNAs include, but are not limitedto miRNA-1, miRNA-122, miR-584, miR-517c, miR-378, miR-520f, miR-142-5p,miR-451, miR-518d, miR-215, miR-376a, miR-133b, miR-367. In certainembodiments, a panel of diagnostic markers includes one or more miRNAsof the miR-17-92 cluster, which are known to be transactivated by c-Myc,such as miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a-1.

Exosomal biomarkers associated with renal tissues and renal diseases areof particular interest in view of being derived from renal epithelialcells and their ready detection in exosomes isolated from urine.Exemplary exosomal biomarkers from normal urine include apicaltransporters present in each renal tubule segment, including theproximal tubule (sodium-hydrogen exchanger 3, sodium-glucoseco-transporter 1 and 2, and aquaporin-1 (AQP1)), the thick ascendinglimb (sodium-potassium-chloride co-transporter 2 (NKCC2)), the distalconvoluted tubule (thiazide-sensitive Na—Cl co-transporter (NCC)), andconnecting tubule/collecting duct (AQP2, rhesus blood group Cglycoprotein (RhCG, an ammonia channel), B1 subunit of vacuolarH⁺-ATPase, and pendrin); hepatocyte growth factor-regulated tyrosinekinase substrate, tumor susceptibility gene 101, vacuolar proteinsorting 28 isoform 1, vacuolar protein sorting 28 isoform 2, vacuolarprotein sorting 37B, vacuolar protein sorting 37C, EAP25, EAP45, EAP30,CHMP2A, CHMP2B, CHMP3, CHMP4B, CHMP5, CHMP1A, CHMP1B, CHMP6, vacuolarprotein sorting factor 4A, and vacuolar protein sorting factor 4B. Adatabase of urinary exosome proteins (and their sequences) from healthyhuman volunteers based on published and unpublished protein massspectrometry data from the NHLBI Laboratory of Kidney and ElectrolyteMetabolism is publicly available atdir.nhlbi.nih.gov/papers/lkem/exosome/.

Detection of Biomarkers

The step of detecting biomarkers in the exosomes may be carried outusing any methodology suitable for isolating and/or detecting aninfectious agent-associated biomarker, including but not limited to massspectrometry (MS), including liquid chromatography-tandem massspectrometry (LC-MS/MS), surface enhanced laser desorption/ionizationtime of flight mass spectrometry (SELDI-TOF-MS), high-pressure liquidchromatography-mass spectrometry (HPLC-MS) and fast protein liquidchromatography (FPLC); Fluorescence-activated cell sorter (FACS)analysis, Western blot, enzyme-linked immunosorbent assay (ELISA),including sandwich ELISA, de novo protein sequencing (e.g., viaLC-MS/MS), nucleotide sequencing, PCR, quantitative PCR (qPCR) orreal-time PCT, RT-PCR, qRT-PCR. antibody array, test strips,one-dimensional and two-dimensional electrophoretic gel analysis andcombinations thereof.

In certain preferred embodiments, a sample from a subject can becontacted with an antibody that specifically binds an infectiousagent-associated biomarker. Optionally, the antibody can be fixed to asolid support to facilitate washing and subsequent isolation of thecomplex, prior to contacting the antibody with a sample. Examples ofsolid supports include glass or plastic in the form of a microtiterplate, a stick, a bead, or a microbead. Examples of solid supportsencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, silicones, and plastics such as polystyrene,polypropylene and polyvinyl alcohol. The sample can be diluted with asuitable diluant or eluant before contacting the sample to the antibody.

After incubating the sample with antibodies, the mixture is washed andthe antibody-biomarker complex formed can be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.This detection reagent may be a second antibody which is labeled with adetectable label, for example. Exemplary detectable labels includemagnetic beads (e.g., DYNABEADS™), fluorescent dyes, radiolabels,enzymes (for example, horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads. Alternatively, thebiomarker in the sample can be detected using an indirect assay,wherein, for example, a second, labeled antibody is used to detect boundbiomarker-specific antibody, and/or in a competition or inhibition assaywherein, for example, a monoclonal antibody which binds to a distinctepitope of the marker is incubated simultaneously with the mixture.

Immunoassays can be used to determine presence or absence of infectiousagent-associated biomarker(s) in a sample as well as the quantity of thebiomarker(s) in the sample. If a marker is present in the sample, itwill form an antibody-marker complex with an antibody that specificallybinds the marker under suitable incubation conditions described above.The amount of an antibody-marker complex can be determined by comparingto a standard. A standard can be a known compound or another proteinknown to be present in a sample, for example. As noted above, the testamount of marker need not be measured in absolute units, as long as theunit of measurement can be compared to a control.

The term “antibodies” as used herein includes both polyclonal andmonoclonal antibodies. In addition to intact immunoglobulin molecules,also included in the term “antibodies” are fragments or polymers ofthose immunoglobulin molecules, and human or humanized versions ofimmunoglobulin molecules or fragments thereof, as long as they arechosen for their ability to interact with an infectious agent-associatedbiomarker. The antibodies can be tested for their desired activity usingthe in vitro assays described herein, or by analogous methods, afterwhich their in vivo therapeutic and/or prophylactic activities aretested according to known clinical testing methods.

Exemplary antibody or antibody derived fragments may include any memberof the group consisting of: IgG, antibody variable region; isolated CDRregion; single chain Fv molecule (scFv) comprising a VH and VL domainlinked by a peptide linker allowing for association between the twodomains to form an antigen binding site; bispecific scFv dimer; minibodycomprising a scFv joined to a CH3 domain; diabody (dAb) fragment; singlechain dAb fragment consisting of a VH or a VL domain; Fab fragmentconsisting of VL, VH, CL and CH1 domains; Fab′ fragment, which differsfrom a Fab fragment by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain, including one or more cysteinesfrom the antibody hinge region; Fab′-SH fragment, a Fab′ fragment inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup; F(ab′)₂, bivalent fragment comprising two linked Fab fragments;Fd fragment consisting of VH and CH1 domains; derivatives thereof; andany other antibody fragment(s) retaining antigen-binding function. Fv,scFv, or diabody molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains. When usingantibody-derived fragments, any or all of the targeting domains thereinand/or Fc regions may be “humanized” using methodologies well known tothose of skill in the art. In some embodiments, the infectious-agentassociated antibody is modified to remove the Fc region.

In certain embodiments, the infectious agent-associated biomarkers aredetected using enzyme-linked immunosorbent assay (ELISA) which istypically carried out using antibody coated assay plate or wells.Commonly used ELISA assay employs either a sandwich immunoassay or acompetitive binding immunoassay.

A sandwich ELISA may be used to capture, detect, characterize andquantify exosomes from small volumes of body fluids. A sandwich ELISAemploys two antibodies, which bind to different sites on the antigen orligand. The primary antibody, which is highly specific for the antigen,is attached to a solid surface. The antigen is then added followed byaddition of a second antibody referred to as the detection antibody. Thedetection antibody binds the antigen to a different epitope than theprimary antibody. Each of these antibodies may be directed to adifferent exosomal marker epitope on the same or different exosomalproteins, whereby the primary antibody captures the exosome or proteintherefrom via the first exosomal protein and the detection antibodyfacilitates quantitation of a exosomal protein bound thereto. As aresult, the exosome or protein therefrom is “sandwiched” between the twoantibodies. A sandwich ELISA may also use lectins to capture exosomes.Lectins have a particular affinity for glycan markers, such asglycoproteins, which are often present in tumor cell-derived exosomes,such as HCC-derived exosomes.

Non-limiting examples of lectins for immobilization on a substrateinclude Lens culinaris agglutin (LCA), Galanthus nivalis lectin (GNA),Narcissus pseudonarcissus lectin (NPL), Allium sativum lectin (ASA),Lens culinaris lectin (LCH), Sambucus nigra lectin (SNA), Maackiaamurensis lectin (MAL), Concanavalin A (Con A), Aleuria aurantia lectin(AAL), Lotus tetragonolobus lectin (LTL), Naja mossambica lectin (NML),Dolichos biflorus agglutinin (DBA), Helix aspersa lectin (HAL),Psophocarpus tetragonolobus lectin II (PTL II), Wisteria floribundalectin (WFL), Erythrina cristagalli lectin (ECL), Griffoniasimplicifolia lectin II (GSL II) and Phaseolus vulgaris leucoagglutinin(PHA-L).

AFP-L3, an isoform of alpha-fetoprotein, is the major glycoform in theserum of HCC patients and is known to bind LCA. For diagnosis of HCC,the AFP-L3 marker may be detected in combination with other AFPglycoforms, including AFP-P4, AFP-P5 (E-PHA), and monosialylated AFP. Incontrast, the L1 isoform of AFP (AFP-L1) may be used to diagnose anon-HCC inflammation of liver disease condition. In certain preferredembodiments, LCA lectin is used to bind exosomes in bodily fluids fromHCC subjects.

The binding affinity for the antigen (via antibodies or lectins) isusually the main determinant of immunoassay sensitivity. As the antigenconcentration increases the amount of binding agent bound increasesleading to a higher measured response. The standard curve of asandwich-binding assay has a positive slope. To quantify the extent ofbinding different reporters can be used. Typically an enzyme is attachedto the secondary antibody which must be generated in a different speciesthan primary antibodies (i.e., if the primary antibody is a rabbitantibody than the secondary antibody would be an anti-rabbit from goat,chicken, etc., but not rabbit). The substrate for the enzyme is added tothe reaction that forms a colorimetric readout as the detection signal.The signal generated is proportional to the amount of target antigenpresent in the sample. The antibody linked reporter used to measure thebinding event determines the detection mode. A spectrophotometric platereader may be used for colorimetric detection. Several types ofreporters have been developed in order to increase sensitivity in animmunoassay. For example, chemiluminescent substrates have beendeveloped which further amplify the signal and can be read on aluminescent plate reader. Also, a fluorescent readout may be obtainedwhere the enzyme step of the assay is replaced with a fluorophor taggedantibody. This readout is then measured using a fluorescent platereader.

Biomarker Panels

In certain embodiments, a biomarker panel of at least 2, 3, 4, 5, 10,15, 20, 25, 30, 40 or 50 liver disease markers in microarray may beemployed. In certain embodiments, a biomarker panel assays expression ofbiomarker proteins. In other embodiments, the biomarker panel assaysexpression of biomarker mRNAs or miRNAs. For example, infectiousagent-associated biomarkers may be detected using a biomarker microarraypanel containing immobilized infectious agent-associatedbiomarker-specific antibodies on a substrate surface. The microarray canbe used in a “sandwich” assay in which the antibody on the microarraycaptures an infectious agent-associated biomarker in the test sample andthe captured marker is detected by a labeled secondary antibody thatspecifically binds to the captured marker. In a preferred embodiment,the secondary antibody is biotinylated or enzyme-labeled. The detectionis achieved by subsequent incubation with a streptavidin-fluorophoreconjugate (for fluorescence detection) or an enzyme substrate (forcolorimetric detection).

Typically, a microarray assay contains multiple incubation steps,including incubation with the samples and incubation with variousreagents (e.g., primary antibodies, secondary antibodies, reportingreagents, etc.). Repeated washes are also needed between the incubationsteps. In one embodiment, the microarray assays is performed in a fastassay mode that requires only one or two incubations. It is alsoconceivable that the formation of a detectable immune complex (e.g., acaptured infectious agent-associated biomarker/anti-markerantibody/label complex) may be achieved in a single incubation step byexposing the biomarker microarray to a mixture of the sample and all thenecessary reagents. In one embodiment, the primary and secondaryantibodies are the same antibody.

In another embodiment, the biomarker microarray provides a competitiveimmunoassay. Briefly, a microarray comprising immobilized anti-markerantibodies is incubated with a test sample in the presence of a labeledinfectious agent-associated biomarker standard. The labeled infectiousagent-associated biomarker competes with the unlabeled infectiousagent-associated biomarker in the test sample for the binding to theimmobilized antigen-specific antibody. In such a competitive setting, anincreased concentration of the specific infectious agent-associatedbiomarker in the test sample would lead to a decreased binding of thelabeled infectious agent-associated biomarker standard to theimmobilized antibody and hence a reduced signal intensity from thelabel.

In certain embodiments, a diagnosis may include an oligonucleotidemicroarray for detecting and quantitating miRNA expression level(s). Anoligonucleotide microarray consists of an arrayed series of a pluralityof microscopic spots of oligonucleotides, called features, eachcontaining a small amount (typically in the range of picomoles) of aspecific oligonucleotide sequence. The specific oligonucleotide sequencecan be a short section of a gene or other oligonucleotide element thatis used as a probe to hybridize a cDNA or cRNA sample underhigh-stringency conditions. Probe-target hybridization is usuallydetected and quantified by fluorescence-based detection offluorophore-labeled targets to determine relative abundance of nucleicacid sequences in the target. The oligonucleotide probes are typicallyattached to a solid surface by a covalent bond to a chemical matrix (viaepoxy-silane, amino-silane, lysine, polyacrylamide or others). The solidsurface can be glass or a silicon chip or microscopic beads.Oligonucleotide arrays are different from other types of microarray onlyin that they either measure nucleotides or use oligonucleotide as partof its detection system.

A biomarker microarray panel can be processed in manual, semi-automaticor automatic modes. Manual mode refers to manual operations for allassay steps including reagent and sample delivery onto microarrays,sample incubation and microarray washing. Semi-automatic modes refer tomanual operation for sample and reagent delivery onto microarray, whileincubation and washing steps operate automatically. In an automaticmode, three steps (sample/reagent delivery, incubation and washing) canbe controlled by a computer or an integrated breadboard unit with akeypad. For example, the microarray can be processed with aPROTEINARRAY™ Workstation (PerkinElmer Life Sciences, Boston, Mass.) orAssay 1200™. Workstation (Zyomyx, Hayward, Calif.). Scanners byfluorescence, colorimetric and chemiluminescence, can be used to detectmicroarray signals and capture microarray images. Quantitation ofmicroarray-based assays can also be achieved by other means, such asmass spectrometry and surface plasma resonance. Captured microarrayimages can be analyzed by stand-alone image analysis software or withimage acquisition and analysis software package. For example,quantification of an antigen microarray can be achieved with afluorescent PMT-based scanner SCANARRAY™ 3000 (General Scanning,Watertown, Mass.) or colorimetric CCD-based scanner—VisionSpot (AlliedBiotech, Ijamsville, Md.). Typically, the image analysis would includedata acquisition and preparation of assay report with separate softwarepackages. To speed up the whole assay process from capturing an image togenerating an assay report, all the analytical steps including imagecapture, image analysis, and report generation, can be confined inand/or controlled by one software package. Such an unified controlsystem would provide the image analysis and the generation of assayreport in a user-friendly manner.

Exosomes may be isolated by a variety of methodologies, including butnot limited to density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity capture, size-exclusion chromatography, ultracentrifugation,and ultracentrifugation followed by size-exclusion chromatography(UC-SEC), magnetic activated cell sorting (MACS), combination thereof,and the like.

In one embodiment, the isolating step is accomplished by sedimentingexosomes in a bodily fluid sample via centrifugation. The sedimentedexosomes are washed and resuspended at a proper concentration forfurther analysis. In certain embodiments, the sample may be centrifugedat 100,000×g or above for 10-120 or 60-120 minutes to sediment theexosomes.

In certain embodiments, the exosomes in the bodily fluid sample areprecipitated by a two-step centrifugation process that includes a low gforce centrifugation to remove calls and other large particles in theurine and a high g force centrifugation to precipitate the exosomes. Inone embodiment, the sample is first centrifuged at 5,000-25,000×g for5-30 minutes. The supernatant is then transferred to another tube and iscentrifuged again at 100,000×g or above for 30-120 minutes to sedimentthe exosomes. In a preferred embodiment, the bodily fluid sample isfirst centrifuged at 20,000-22,000×g for 10-20 minutes. The supernatantis then transferred to another tube and is centrifuged again at100,000×g for 30-90 minutes to sediment the exosomes. The sedimentedexosomes are then resuspended in a liquid medium for further analysis.

The liquid medium can be isotonic, hypotonic, or hypertonic. In certainembodiments, the liquid medium contains a buffer and/or at least onesalt or a combination of salts. Buffers can maintain pH within aparticular range, for example, between 1 and 12, and are also referredto as pH stabilizing agents. More typically, pH will range within aboutpH 5.0 to about pH 12.0. A particular example of a pH stabilizing agentis a zwitterion. Specific non-limiting examples of pH stabilizing agentsinclude Tris(hydroxymethyl)aminomethane hydrochloride (TRIS),N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES),3-(N-morpholino) propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES),N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES),N-[carboxymethyl]-2-aminoethanesulfonic acid (ACES),N-[2-acetamido]-2-iminodiacetic acid (ADA),N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES),N-[2-hydroxyethyl]piperazine-N-[2-hydroxypropoanesulfonic acid](HEPPSO), N-tris[hydroxymethyl]methylglycine (TRICINE),N,N-bis[2-hydroxyethyl]glycine (BICINE),4-(cyclohexylamino)-1-butanesulfonic acid (CABS),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),3-(cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO),2-(cyclohexylamino)ethanesulfonic acid (CHES),N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid) (EPPS),piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES),[(2-hydroxy-1,1-bis[hydroxymethyl]ethyl)amino]-1-propanesulfonic acid(TAPS), N-tris(hydroxymethyl)methyl-4-aminobutane sulfonic acid (TABS),2-amino-2-methyl-1-propanol (AMP),3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid(AMPSO), ethanolamine and 3-amino-1-propanesulfonic acid. Additionalspecific non-limiting examples of pH stabilizing agents includepotassium chloride, citric acid, potassium hydrogenphthalate, boricacid, potassium dihydrogenphosphate, diethanolamine, sodium citrate,sodium dihydrogenphosphate, sodium acetate, sodium carbonate, sodiumtetraborate, cacodylic acid, imidazole, 2-Amino-2-methyl-1-propanediol,tricine, Gly-Gly, bicine, and a phosphate buffer (e.g., sodium phosphateor sodium-potassium phosphate, among others).

Buffers or pH stabilizing agents are typically used in a range of about0.1 mM to about 500 mM, in a range of about 0.5 mM to about 100 mM, in arange of about 0.5 mM to about 50 mM, in a range of about 1 mM to about25 mM, or in a range of about 1 mM to about 10 mM. More particularly,buffers can have a concentration of about (i.e., within 10% of) 1 mM, 2mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM.

The liquid medium may further contain a chelating agent. Chelatingagents typically form multiple bonds with metal ions, and aremultidentate ligands that can sequester metals. Metal sequestration canin turn reduce or prevent microbial growth or degradation ofbiomolecules (e.g., peptide or nucleic acid), which in turn can improvepreservation of biomolecules absorbed to a substrate. Specificnon-limiting examples of chelating agents include EDTA(ethylenediamine-tetraacetic acid), EGTA(ethyleneglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid),GEDTA (glycoletherdiaminetetraacetic acid), HEDTA(N-(2-hydroxyethylethylenediamine-N,N′,N′-triacetic acid), NTA(nitrilotriacetic acid), salicylic acid, triethanolamine and porphine.Typical concentrations of chelating agents are in a range of about 0.1mM to about 100 mM, in a range of about 0.5 mM to about 50 mM, or in arange of about 1 mM to about 10 mM.

The liquid medium may also contain a denaturing agent. Denaturing agentsand detergents typically form a chemical bridge between hydrophobic andhydrophilic environments, which in turn disrupt or diminish thehydrophobic forces required to maintain native protein structure.Particular non-limiting chemical classes of denaturing agents anddetergents include anionic surfactants, nonionic surfactants, cationicsurfactants and ampholytic surfactants. Specific non-limiting examplesof detergents include guanidinium thiocyanate, sodium dodecyl sulfate,sodium lauryl sulfate, NP40, Triton X-100, Tween, sodium cholate, sodiumdeoxycholate, benzethonium chloride, CTAB (cetyltrimethylammoniumbromide), hexadecyltrimethylammonium bromide, andN,N-dimethyldecylamine-N-oxide.

The liquid medium may further contain a denaturing agent. Reducingagents and antioxidants typically inhibit microbial growth and reducebiomolecule oxidation. Particular non-limiting classes of such agentsinclude free radical scavenging agents. Specific non-limiting examplesof reducing agents and anti-oxidants include DTT (dithiothreitol),dithioerythritol, urea, uric acid, 2-mercaptoethanol, cysteine, vitaminE, vitamin C, dithionite, thioglycolic acid and pyrosulfite.

The liquid medium may further contain a preservative or stabilizingagent. Preservatives or stabilizing agents can be used if it is desiredto inhibit or delay degradation of an the biomarkers of interest.Specific non-limiting examples of preservatives and stabilizing agentsinclude sodium azide and polyethylene glycol (PEG). Typicalconcentrations of preservatives and stabilizing agents range from about0.05% to about 1%.

The liquid medium may further contain one or more protease inhibitors.Protease inhibitors inhibit peptide degradation. Particular non-limitingclasses of protease inhibitors include reversible or irreversibleinhibitors of substrate (e.g., peptide) binding to the protease.Particular non-limiting classes of protease inhibitors include serineand cysteine protease inhibitors. Specific non-limiting examples ofprotease inhibitors include PMSF, PMSF Plus, APMSF, antithrombin III,amastatin, antipain, aprotinin, bestatin, benzamidine, chymostatin,calpain inhibitor I and II, E-64,3,4-dichloroisocoumarin, DFP,elastatinal, leupeptin, pepstatin, 1,10-phenanthroline, phosphoramidon,TIMP-2, TLCK, TPCK, trypsin inhibitor (soybean or chicken egg white),hirustasin, alpha-2-macroglobulin, 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF) and Kunitz-type protease inhibitors.

In another embodiment, exosomes in a bodily fluid sample are collectedby passing the bodily fluid sample through a filter having a pore sizethat is smaller than the average size of exosomes. The exosomes are thenremoved from the filter and resuspended at a proper concentration forfurther analysis. In certain embodiments, exosomes in the bodily fluidsamples are collected using centrifuge filters with a molecular weightcutoff of 500 kd-50 kd. In one embodiment, exosomes in the bodily fluidsamples are collected using centrifuge filters with a molecular weightcutoff of 100 kd.

In other embodiments, bodily fluids or cell-free supernatants thereofmay be incubated with beads coated with one or more antibodiesrecognizing marker proteins on the surface of exosome particles.Exemplary exosome surface markers include, but are not limited to MEWclass II markers, including those of the HLA DP, DQ and DR haplotypes;CD9, CD63, CD81 and CD82.

For example, exosome surface marker-directed antibodies be attached tomagnetic beads, such as those manufactured by DYNABEADS® (Dynal, Oslo,Norway) for affinity purification of exosomes. More specifically,exosomes having CD63 on their surface may be isolated using antibodycoated magnetic bead particles. DYNABEADS® are super-paramagneticpolystyrene beads which may be conjugated with anti-human CD63 antibody,either directly to the bead surface or via a secondary linker (e.g.,anti-mouse IgG). The beads may be between 1 and 4.5 μmin diameter.

Antibody coated DYNABEADS® may be added to an exosome sample preparedusing a volume-excluding polymer and incubated at 2-8° C. or at roomtemperature from 5 minutes to overnight. DYNABEADS® with bound exosomesmay then be collected using a magnet. The isolated bead-bound exosomesmay then be resuspended in an appropriate buffer, such as phosphatebuffered saline, and used for downstream analysis (qRT-PCR, sequencing,Westerns, flow cytometry, etc.). Similar protocols may be used for anyother exosome surface marker for which an antibody or other specificligand is available. Indirect binding methods such as those usingbiotin-avidin may also be used.

Once an isolated exosome sample has been prepared, the contents of theexosome may be analyzed directly or further extracted for additionalstudy and characterization. Biological material which may be extractedfrom exosomes includes proteins, peptides, RNA and DNA, lipids. Forexample, the MIRVANA™ PARIS™ Kit (AM1556, Life Technologies) may be usedto recover native protein and RNA from exosomes samples, including smallRNAs, such as miRNAs, snRNAs, and snoRNAs.

Total RNA may be extracted using acid-phenol:chloroform extraction. RNAmay then be purified using a glass-fiber filter under conditions thatrecover small-RNA containing total RNA, or that separate small RNAspecies less than 200 nucleotides in length from longer RNA species suchas mRNA. Because the RNA is eluted in a small volume, no alcoholprecipitation step may be required for isolation of the RNA.

Kits

Another aspect of the present application relates to a kit fordiagnosing an infectious agent-associated disease or monitoring theprogress of an infectious agent-associated disease in a subject. The kitcontains one or more reagents for preparing an exosome preparation; oneor more infectious agent-associated biomarker binding agents selectivefor one or more infectious disease(s) or infectious diseasecondition(s); one or more infectious agent-associated biomarkerstandard(s), and one or more detection reagents for detecting binding ofthe one or more biomarker binding agents(s) to one or more infectiousagent-associated biomarker(s).

The detection reagents for detecting binding of the one or morebiomarker binding agents(s) to one or more infectious agent-associatedbiomarker(s) may include antibody reagents, including horseradishperoxidase (HRP)-antibody conjugates and the like for detection andquantitation of protein levels, as well amplification primers oroligonucleotide probes for detection and quantitation of infectiousagent associated genomic nucleic acids, mRNA levels and miRNAs.

In some embodiments, the kit is a hepatitis virus infection detectionkit comprising one or more reagents for preparing an exosomepreparation; one or more hepatitis virus-associated biomarker bindingagents; one or more hepatitis virus-associated biomarker standards, andone or more detection reagents for detecting binding of the one or morehepatitis virus-associated biomarker binding agents to one or morehepatitis virus-associated biomarkers. In a related embodiment, the oneor more hepatitis virus-associated biomarker binding agents bind to oneor more hepatitis virus biomarkers selected from the group consisting ofHep C core antigen, Hep C NS2 protein, Hep C NS3 protein and Hep C NS4protein.

In certain embodiments, the kit comprises one or more centrifuge filtersfor collecting exosomes in the bodily fluid samples. In a relatedembodiment, the one or more centrifuge filters have a molecular weightcutoff of 500 kd-50 kd. In another related embodiment, the one or morecentrifuge filters have a molecular weight cutoff of 100 kd.

The kits described above may additionally include liquids suitable forresuspending exosomes isolated from a bodily fluid sample and one ormore container(s) for collecting a bodily fluid sample and/or acentrifuge filter for isolating exosomes from the bodily fluid sample.Additionally, the kits described above will typically include a label orpackaging insert including a description of the components orinstructions for use. Exemplary instructions include, instructions forcollecting a bodily fluid sample, for harvesting exosomes from thesample, and for detecting an infectious agent-associated biomarker.

The present application is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables, are incorporatedherein by reference.

Example 1: Materials and Methods

Patients

HIV+ patients, at various stages of disease, were recruited for thisstudy from four clinical sites in the Atlanta metropolitan area. Onlythose patients on dialysis were excluded from this study. All sampleswere collected in accordance with protocols approved by theInstitutional Review Board and the Human Subjects Research Committee atMorehouse School of Medicine, and informed consent was obtained from allpatients and healthy volunteers according to the guidelines institutedby the Institutional Review Board. Patients were divided into fivegroups: African American patients with HIV (AA HIV+), white patientswith HIV (White HIV+), patients with HIVAN (HIVAN), African Americanpatients with no HIV but FSGS, and healthy controls. Pertinentinformation was also collected from the medical record of the patients.

Sample Collection and Storage

Urine samples were collected from patients during routine clinicalvisits. Clinical data were obtained from the medical record of thepatients. Urine was collected in sterile containers and transported backto the laboratory. Urinalysis was performed on each specimen using aMULTISTIX® 10 SG Reagent Strip (Bayer Corporation, Elkhart, Ind.) andthe albumin to creatine ratio determined by a Siemens CLINITEK®Microalbumin dipstick (Bayer Corp.). The strips were read on a SiemensCLINITEK STATUS® instrument (Bayer Corp.). Samples were centrifuged at2,000×g for 10 minutes to remove whole cells and sediment. The remainingurine samples were aliquoted into 4 ml volumes and stored at −80° C.until they were analyzed.

Isolation of Exosomes

Two methods were evaluated for the isolation of exosomes, either highspeed ultracentrifugation or ultrafiltration using a molecular weightcutoff filter. For the ultracentrifugation method, 4 ml of urine weretransferred into a polycarbonate centrifuge tube and centrifuged at21,000×g for 15 minutes. The supernatant was removed and againcentrifuged at 100,000×g for 60 minutes to sediment the exosomes. Theexcess urine was decanted and the pellet was reconstituted in 100 μlphosphate buffered saline (PBS) and stored at 4° C. For theultrafiltration method, 4 ml of urine were added to an AMICON® Ultracentrifugal filter device (Ultracel, 100k cutoff, Millipore, Inc.) andcentrifuged at 4,000×g for 20 min in a swinging bucket rotor. Onehundred μl of PBS was used to rinse the filter and dilute the retentate.The protein concentration was determined using the bicinchoninic acidprotein assay (Pierce).

Normal phase chips (PROTEINCHIP® NP20; Ciphergen Biosystems, Fremont,Calif.), that bind proteins through hydrophilic and charged residueswere used for the analysis. Five μl of vesicle preparation was appliedin duplicate to the chip and incubated for 30 minutes in a humidchamber. Chips were washed three times with 5 μl high-performance liquidchromatography (HPLC)-grade water and air dried for 10 minutes.Saturated sinapinic acid (SPA, Ciphergen Biosystems, CA) were preparedin 50% acetonitrile/0.5% trifluoroacetic acid according to manufacturersinstructions. One μl of matrix solution (SPA) to each spot and air-driedand subsequently read with the PROTEINCHIP® Reader II, (CiphergenBiosystems) using the following settings: laser intensity 250; detectorsensitivity 10; high mass 300 Kda, optimized from 3 Kda to 50 Kda. Thedata acquisition method was set to automatic laser adjustment and peakswere auto identified from 3 Kda and 50 Kda.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Collected exosomes were analyzed by LC/MS using an LTQ mass spectrometer(ThermoFinnigan). The pelleted exosomes were first extracted with 2 Dgel loading buffer (Q-biosciences) made fresh the day of analysis. Thesolubilized pellet was then precipitated using four volumes of ice-cold(−20° C.) acetone and incubated overnight at −20° C. The precipitate wascollected by centrifugation at 19,200×g. The pellet was dried andre-dissolved into 50 mM ammonium bicarbonate (AmBIC). The proteinsolution n was first reduced using 2 μl of a 500 mM stock of DTT (Qbiosciences, single use) at 56° C. for 30 minutes. The solution was thenalkylated by adding 2 μl of a 1M stock of iodacetic acid (IAA;Q-biosciences, single use) and incubating at room temperature for 30minutes in the dark. A fresh vial of trypsin (Promega Gold mass specgrade) was diluted 8 μl to 312 μl in 50 mM AmBic and kept on ice. Tenmicroliters of the diluted trypsin was then added to the reaction and itwas incubated at 37° C. for 4 hours with shaking. Then 50 μl of 0.5%formic acid was added and the mixture was either directly analyzed orstored at −20° C. for analysis. Ten microliters of sample was injectedusing an automated sampler onto a captrap (Michrom) C18 peptide trap ata flow of 10 μl per min. After 10 min the flow was switched to a 0.5mm×50 micron C18 column (Michrom). Peptides were eluted using a lineargradient of 5-40% acetonitrile in water over 50 min. The eluted peptideswere directly introduced into an LTQ mass spectrometer using microsprayionization (Michrom ADVANCE™) at a flow rate of approximately 3 μl permin. Samples were analyzed using Excalibur 2.2 software set to analyzeions in a data dependent scanning mode. A precursor scan was followed bydata dependent scans of the three most intense ions. Files were searchedagainst a subset of the NR database that included Human and HIV proteinsusing BioWorks 3.1 (ThermoFinnigan). The threshold for DTA generationwas set at 200 and the tolerance for peptides was set at 0.5 Da andproteins at 1.0 Da. Initial protein identification lists were generatedusing consensus scores of >10.0 and Xcorr scores >1.0.

Electrophoresis and Western Blot

SDS PAGE electrophoresis. Samples were heated at 85° C. for 2 minutes ina Tris-Glycine SDS sample reducing buffer and loaded in a 4-12%CRITERION™ XT Bis-Tris precast acrylamide gel (BioRad, Hercules,Calif.). Approximately 200 ng of sample were loaded into each well.Controls consisted of recombinant HIV Nef (gift of Dr. Andrea Raymond)and HIV recombinant p24 (Immunodiagnostics, Inc.) that were loaded at 30to 40 ng per well. The gels were stained using Gel Code Blue (Pierce,Inc.) or the proteins were transferred to PVDF membrane (Immobilon-P,Millipore Corp, Billerica, Mass.) for western blot analysis. The SNAPID® system (Millipore, Corp) was used for the western blot analysis forthe presence of either HIV Nef or HIV proteins. HIV Nef identificationwas performed using a monoclonal mouse anti-HIV Nef monoclonal antibody(1:1500, Chemicon Int., CA) and a secondary antibody, goat anti-mouseIgG (H+L) peroxidase conjugated antibody (1:15,000, JacksonImmunoresearch, West Grove, Pa.). HIV proteins were detected usingpooled human HIV+ serum (1:15,000) as the primary antibody and a goatanti-human IgG (H+L) peroxidase conjugated antibody (1:15,000, JacksonImmunoresearch). The membrane was incubated with a chemiluminescentsubstrate (SuperSignal West Femto Maximum, Pierce, Inc.) and exposed toX-ray film (CL Xposuro CL-XPOSURE™, Kodak) and developed.

Transmission Electron Microscopy

Samples were fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer for2 hours at 4 C followed by 2 washes with 0.1M cacodylate buffer, 5minutes each. The samples were fixed again with 1% osmium tetroxide in0.1M cacodylate buffer for 1 hour at 4° C. followed by 2 washes with thecacodylate buffer and 3 washes with deionized water, 5 minutes each.Thin sections were cut, stained with 0.5% aqueous uranyl acetate for 2hours at room temperature, and viewed with a JEOL 1200EX transmissionelectron microscope.

Example 2: Isolation of Urinary Vesicles, Ultracentrifugation VersusUltrafiltration

Vesicles from the urine of six (6) different HIV+ patients were isolatedby ultracentrifugation or ultrafiltration to determine which of the twomethods yielded the greatest amount of protein. The ultrafiltrationmethod consistently isolated more protein (2930 μg median) than theultracentrifugation method (591 median).

Example 3: SELDI-TOF-MS Analysis of Hiv-Associated Biomarkers in UrinaryExosomes from Patients

FIG. 1 is a flow chart showing an embodiment of an exemplary method fordetecting HIV-infection or monitoring the progress of HIV-infection in asubject using a urine sample from the subject. Urinary exosomes frompatients of various groups were analyzed for the presence ofHIV-associated biomarkers by SELDI-TOF-MS. The results are confirmed byLC-MS/MS. Spectrum of SELDI-TOF-MS from representative patients is shownin FIGS. 2-6. Table 1 summarizes HIV-associated proteins that weredetected by SELDI-MS and confirmed by LC-MS/MS in different test groups.Table 2 summarizes the urine protein profiles in individual patient.

TABLE 1 HIV-associated proteins detected in urine samples by SELDI-MSPatient MW Protein HIVAN 10,585 HIV envelope gp; HIV Protease 23,546 HIVenvelope gp; HIV Nef; HIV Vif 33,464 HIV protein gp; mu A03009B12RikProtein 45,632 HIV envelope gp; HIV pol protein 66,587 HIV envelope gp;HIV Nef; PgD synthase 78,942 Unknown AA HIV 23,684 HIV envelope gp; HIVNef; PgD synthase 83,256 Unknown FSGS 66,533 Unknown White HIV 23,935

TABLE 2 Urine protein profiles in individual patient ID Diagnosis RaceCD4 VL Nef Gag Pol Protease Rev RT Tat Vif p1 p24 P17 poly 22 HIVAN AA XX X X X X X X X X 27 HIV/AIDS AA 134 <50 X 28 HIV/AIDS AA 134 19,800 X XX X 30 HIV/AIDS AA <20 <10,000 X X X X X X X X X X 41 HIV AA 440 29,187X X X 46 HIV AA 689 <50 X X X X X 62 HIV AA 232 <50 X 63 HIV/AIDS AA 832,023 X 70 HIV AA 990 <50 X 104 HIV AA 313 77 X X 111 HIV/AIDS AA 182<50 X X 112 HIV AA 584 <200 X X 48 HIV W 454 52000 X X X X 86 HIV W 1642<75 X X X X 103 HIV W 560 150 X X X 106 HIV W 302 <50 108 HIV W 653 <50X X X 110 HIV W 379 <50 X XTransmission Electron Microscopy (TEM)

TEM was used to visualize the patients' vesicles from urine. Exosomeswere isolated from 4 ml of urine, fixed and embedded for TEM. The figureshows distribution of vesicles in: A) HIVAN; B) focal segmentalglomerulosclerosis; C) AA HIV+; and D) white HIV+; E) AA HIV negative.HIVAN, FSGS and AA HIV+ patients clearly have a higher population ofvesicles compared to white HIV+ patients and AA normal patients.

Ingenuity Pathways Analysis

As shown in Examples 1 and 2, the SELDI-TOF-MS peaks of AA HIV+ patientsexhibited exceedingly similar protein patterns to those of HIVANpatients and slightly similar patterns to FSGS patients, suggesting thatthe AA HIV+ patients whose peaks were similar to those of HIVAN may bepredisposed to developing HIVAN. The baseline protein value (30-2000mg/dl) for the FSGS patients and AA HIV+ patients was in the same range.Similar to the protein values for HIVANb, but unlike FSGS patients, theproteins detected in AA HIV+ patients were analogous to those of HIVANpatients. This underscores the significance that HIV infection, with orwithout the presence of renal disease, is still largely responsible fordevelopment of HIVAN; and a prior condition of renal insufficiencybefore HIV infection is not a necessary prerequisite for the developmentof HIVAN.

Unlike AA HIV+ patients, protein profiles of white HIV+ patients were astark contrast to the protein profiles of HIVAN patients. It suggeststhat factors other than simple infection of renal cells or theinfiltration of infected immune cells in renal tissue probably mediatethe expression of nephropathy. The number of AA HIV+ and HIVAN (12/15)patients that had detectable Nef using LC-MS/MS piggybacks on theearlier assertion about the similarities between the kidney pathologiesof transgenic mice expressing Nef and HIVAN patients, hinting that Nefmay be involved in causing kidney damage to HIV patients. This mayexplicate the relationship, if any, between the similarity in the kidneypathology of transgenic mice expressing Nef and HIVAN patients, and thesimilarity between the expression of Nef in the protein profiles of AAHIV+ and HIVAN patients. It may also shed additional insight as to whatrole Nef plays in the pathogenesis of HIVAN. HIV envelope gp was alsodetected by LC-MS/MS in HIVAN and AA HIV+ patients. Although local HIVinfection of the kidney may have implications beyond development ofHIVAN, with kidney serving as a potential viral reservoir, a corollarywould be that some of these viruses in the reservoir would find theirway in the urine.

The transmission electron microscopy (FIG. 7) shown in the urine of AAHIV+, FSGS and HIVAN patients' conspicuous vesicles were not evident inthe urine of white HIV+ and AA normal patients. The HIVAN exosomesolution was diluted 10 fold because the initial visualization expressedan exceedingly dense population of exosomes that was difficult tovisualize, suggesting that HIVAN patients may be producing vesicles atan increasing rate than all the other patient groups. The HIV-associatedkidney damage may responsible for this marked increase in exosomeexcretion in AA HIV+ and HIVAN patients.

Example 4: Western Blot Analysis, Validation of the Presence of HIV NEFand Other HIV Proteins

Urinary vesicle samples from fourteen (14) HIV+AA and nine (9) HIV+white patients were isolated using ultrafiltration and analyzed for thepresence of HIV Nef and other HIV proteins using western blot analysis.All the HIV+AA samples were positive for HIV Nef by western blot,although HIV Nef was not detected in sample 41 by mass spectrometry(FIG. 8). This discrepancy could be caused by the isolation methodutilized for the mass spectrometry analysis, which wasultracentrifugation, and yields less protein. HIV Nef was onlyidentified in four (4) HIV+ white patients while mass spectrometryidentified three (3) samples without HIV Nef. All HIV+ patients haddetectable HIV proteins by western blots, but had varying kinds andamounts (FIG. 8).

Example 5: Sandwich ELISA Analysis of HBV Biomarkers in Urinary Exosomesfrom Patients

Exosome ELISA

The Nunc 96 strip well ELISA plates coated with 100 μl of μg/ml GNA andblocked with Blocking Buffer were washed 1× with 250 μl of Wash Buffer(0.1% Tween-20 in PBS). Positive controls (mannan from S. cerevisiae)and samples were added to the plates in the amount of 100 μl/well andincubated for 1 hour at room temperature. The plates were washed lx with250 μl of Wash Buffer. 100 μl of HRP labeled GNA (1 μg/ml) or anantibody specific to a marker on the exosome of interest were added toeach well and incubated for 1 hour at room temperature. The plates werewashed 4× with 250 μl of Wash Buffer. 100 μl of Tetramethylbenzidine(TMB) were added to each well and incubated for 30 minutes at roomtemperature or until the Blank well begins to show color. The reactionwas stopped by adding 100 μl of 1M H2504 to each well. The plates wereread in an ELISA plate reader at 450 nm. Serial dilution of samples orantibodies was made with Diluent Buffer (1% BSA, 0.1% Tween-20 in PBS).Marker specific-antibodies include the following:

Anti-Hepatitis C NS4b antigen antibody, mouse monoclonal (MA1-7358,Thermo Scientific);

Anti-Hepatitis C NS3 antigen antibody, mouse monoclonal (MA1-7357,Thermo Scientific);

Anti-Hepatitis C Core antigen antibody, mouse monoclonal (ab2582,Abcam); Anti-Hepatitis C Core antigen antibody, mouse monoclonal(MA1-080, Thermo Scientific);

Anti-Hepatitis C NS3 antigen antibody, mouse monoclonal (MA1-21376,Thermo Scientific);

Anti-Hepatitis C NS4 antigen antibody, mouse monoclonal (MA1-91550,Thermo Scientific);

Anti-Hepatitis C NS5a antigen antibody, mouse monoclonal (MA1-7368,Thermo Scientific);

Anti-Hepatitis A virus antibody, goat polyclonal (LS-C103171, LifespanBiosciences);

Anti-Hepatitis B virus antibody, goat polyclonal (LS-05624, LifespanBiosciences);

Anti-ALG6 (alpha-1,3-glucosyltransferase or Dolichyl pyrophosphateMan9GlcNAc2 alpha-1,3-glucosyltransferase) antibody mouse monoclonal,recombinant fragment: SYSGAGKPPM FGDYEAQRHW QEITFNLPVK QWYFNSSDNNLQYWGLDYPP LTAYHSLLCA YVAKFINPDW IALHTSRGYE SQAHKLFIVIRT (SEQ ID NO: 1),corresponding to amino acids 25-115 of Human ALG6 (ab57112, Abcam);

Anti-ALG6 antibody rabbit polyclonal, Synthetic peptide corresponding toa region within N terminal amino acids 36-85 (GDYEAQRHWQ EITFNLPVKQWYFNSSDNNL QYWGLDYPPL TAYHSLLCAY, SEQ ID NO: 2) of human ALG6(NP_037471) (ab80873, Abcam); and

Anti-ALG3 (alpha-1,3-mannosyltransferase orDolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase)antibody—rabbit polyclonal, Recombinant fragment, corresponding to aminoacids 69-122 of Human ALG3 (ab151211, Abcam).

Example 6: Sandwich ELISA Analysis of HBV Biomarkers in Urinary Exosomesfrom Patients

Urinary exosomes isolated from hepatitis patients and normal controlswere analyzed for the presence of hepatitis A virus (HAV), hepatitis Bvirus (HBV) and hepatitis C virus (HCV)-associated biomarkers by exosomeELISA.

FIG. 9 shows the receiver operating characteristic (ROC) curve fordiagnosis of hepatitis B using anti-Hepatitis B antibody in the exosomeELISA.

FIG. 10 is a ROC curve for diagnosis of hepatitis C using HCV coreantigen and HCV NS3 protein as the biomarkers for hepatitis C in theexosome ELISA.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following embodiments. Theembodiments are intended to cover the components and steps in anysequence which is effective to meet the objectives there intended,unless the context specifically indicates the contrary.

What is claimed is:
 1. A method of detecting a hepatitisvirus-associated biomarker in a subject, comprising the steps of:contacting a urine sample from the subject with one or more hepatitisvirus-associated biomarker binding agent(s) that bind to hepatitis Cvirus (HCV), and detecting binding between the one or more hepatitisvirus-associated biomarker binding agent(s) and the hepatitisvirus-associated biomarker, wherein the hepatitis virus-associatedbiomarker is present in exosomes of the urine sample, wherein thehepatitis C virus (HCV) biomarker is a hepatitis C virus (HCV) biomarkerprotein selected from the group consisting of HCV core antigen, HCV E1protein, HCV E2 protein, HCV p7 protein, HCV NS2 protein, HCV NS3protein, HCV NS4a protein, HCV NS4b protein, HCV NS5a protein and HCVNS4b protein.
 2. The method of claim 1, further comprising the step ofisolating exosomes from the urine sample to produce isolated exosomes.3. The method of claim 2, wherein the isolated exosomes comprise wholeexosomes.
 4. The method of claim 1, further comprising the step of:contacting the urine sample with one or more detection reagents capableof detecting one or more liver disease-associated biomarker(s), whereinsaid detection reagents are selected from the group consisting of one orinure reagents for reverse transcription of RNAs, one or more PCRreagents for amplification and detection of genomic nucleic acids ormRNAs, and one or more oligonucleotides for detecting miRNAs.
 5. Themethod of claim 1, further comprising the step of: detecting liverdamage and/or hepatoeellular carcinoma using markers selected from thegroup consisting of CD10, CD26, CD81, aspartate aminotransferase (AST),alanine aminotransferase (ALT), α-fetoprotein (AFP) and AFP isoformsAFP-L1 AFP-L2, AFP-L3 AFP-P4, AFP-P5 (E-PHA), and monosialylated AFP;des-carboxyprothrombin (DCP), α-1-fucosidase (AFU), γ-glutamyltransferase, glypican-3 GPC-3), squamous cell carcinoma antigen (SCCA),golgi protein 73 (GP73) and mucin 1 (MUC-1), 14-3-3 gamma,gamma-glutamyl transferase, protein C (PROC), retinal binding protein 4(RBP4)alpha-1-B glycoprotein (A1BG), alpha-1-acid glycoprotein (AGP),Mac-2-binding protein (M2BP), complement Factor H (CFH) and insulin-likegrowth factor binding protein acid labeled subunit (IGFALS).
 6. Themethod of claim 2, wherein the exosomes are isolated by differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,size-exclusion chromatography, ultracentrifugation, magnetic activatedcell sorting (MACS) or a combination thereof.
 7. The method of claim 1,further comprising the step of administering a therapeutic drug forhepatitis.